PROJECT SUMMARY/ABSTRACT There is a fundamental gap in understanding how peptidoglycan (PG) is biosynthesized in a spatially coordinated fashion to support the polar growth of Mycobacterium tuberculosis (Mtb). This gap represents an important problem because the elongation of the cell envelope, an essential process of bacterial growth, is incomprehensive without understanding the precise mechanism of spatially coordinated PG precursor biosynthesis, transport and cell wall integration. The intracellular membrane domain (IMD) is a discrete area of the plasma membrane (PM) particularly enriched in the subpolar region of actively growing mycobacterial cells. The long-term goal is to understand the role of PM partitioning in mycobacterial physiology and to identify vulnerabilities in this process. As the next step to achieve this goal, the overall objective of this proposal is to gain the fundamental insights into the spatial compartmentalization of PG assembly, so that the IMD can be evaluated as a target for inhibiting the assembly of the PG layer. The central hypothesis is that the IMD is a region of the PM where polyprenol-linked PG precursors are synthesized. The rationale is that characterizing the PM partitioning of the PG biosynthesis will lay the foundation for understanding the role of the IMD in the cell wall elongation, thereby beginning to understand how the robust pathogen Mtb produces and maintains its highly complex cell wall. Guided by published studies and preliminary data from the applicant?s laboratories, this hypothesis will be tested by pursuing two specific aims: 1) Determine the subcellular localization of proteins that synthesize, transport and polymerize PG precursors; 2) Determine the localized production of polyprenol-linked PG precursors and PG polymer. Under the first aim, the working hypothesis, that PG biosynthetic enzymes are spatially and biochemically segregated in the PM, will be addressed by quantitative and super resolution microscopy and by subcellular fractionation. Under the second aim, the working hypothesis, that precursors and polymerized PG are in distinct PM regions, will be determined by bioorthogonal metabolic labeling of PG for microscopic detection and in vitro biotinylation of PG precursors for biochemical detection. The project is innovative because it combines synergistic expertise of two laboratories to dissect the role of compartmentalized PM in mycobacterial PG synthesis, a substantive departure from the status quo in both concept and execution. The proposed research is significant because it reveals whether PM partitioning organizes PG synthesis, an established drug target of high clinical significance. The key data obtained from this study will form the basis to further investigate the role of the IMD in cell wall elongation of Mtb, enhancing our evaluation of IMD disruption as a new route for inhibiting PG synthesis and mycobacterial cell growth. !